Oral vaccinia formulation

ABSTRACT

This invention relates to methods and systems for generating a safe and effective oral smallpox vaccine for humans using a genetically defective strain of vaccinia virus to confer immunity following oral delivery of the vaccine. This invention is one that expands on current use of vaccinia virus propagation developed for gene therapy applications, and pharmaceuticals and nutraceuticals packaging and formulation technologies. The vaccine invention can be delivered as a live virus with the ability to express viral proteins but unable to achieve complete, lytic virus replication, or it may be derived from such a virus, contain additional immunogens, or be delivered as viral antigens. Furthermore, the invention establishes innovative methods for formulation and packaging and for preclinical testing of the vaccine invention for safety, efficacy and potency with the use of human intestinal and other test cells and diagnostic test systems and kits.

BACKGROUND AND SIGNIFICANCE OF THE INVENTION

Currently, international concern is heightened regarding the potentialuse of smallpox (variola) virus as a bioterrorism agent [1],[2]. Thisconcern has increased since the tragic events around September 11^(th)and the fall of 2001, particularly since the virus has been eradicated[3]. Thus, the recommendations developed by key advisory groups, such asthe CDC Advisory Committee on Immunization Practices (ACIP) regardingvaccinia (smallpox) vaccine and the potential use of smallpox virus as abiological weapon (Modlin, 2001; see www.cdc.gov) are important guidancedocuments. Recommendations from the ACIP included the need to developnew vaccines, particularly a reformulated vaccine produced using cellculture techniques, and better research reagents and methods for therapybased on more modern technologies. Additional recommendations regardingvaccination of persons at various levels of risk, including acute orworkplace exposure to infectious or highly attenuated strains, have beenrevised as part of those recommendations.

The degree to which the individuals who received the original smallpoxvaccinations are protected is still a matter of conjecture and debate[3-7]. It is well known that long-lasting protective immune responsesagainst vaccinia virus have been historically documented [3]. CytotoxicT lymphocytes (CTLs; CD4+ and CD8+) are generated following immunizationand memory T cells can be re-stimulated many years later by a variety ofin vitro methods [6], and kinetics of antibody formation uponre-vaccination have been defined [8].

As reported by the ACIP and other sources (e.g., see www.cdc.gov),Dryvax,® the vaccinia (smallpox) vaccine currently licensed in theUnited States, is a lyophilized, live-virus preparation of infectiousvaccinia virus (Wyeth Laboratories, Inc., Marietta, Pa.). The vacciniavaccine does not contain smallpox (variola) virus. Previously, thevaccine had been prepared from calf lymph with a seed virus derived fromthe New York City Board of Health (NYCBOH, 9) strain of vaccinia virusand has a concentration of about 10⁸ pock-forming units (PFU)/ml.Vaccine was administered by using the multiple-puncture technique with abifurcated needle. Although generally not life-threatening, there aresome side effects in a subset of immunized individuals, particularlythose who are immunosuppressed. Current stockpiles of vaccine kept atthe CDC are inadequate, even when only high-risk for exposureindividuals (e.g., military personnel and “first responders” ) aretargeted for potential immunization with dilute vaccine, butparticularly so in the case of a national emergency. Because the vaccinetechnology for vaccine production and immunization against smallpox isvery old and stockpiles are inadequate in light of potentialbioterrorism with smallpox, there is a need for better vaccines,state-of-the art methods of large-scale vaccine production and safermethods for re-immunization as well as de novo immunization ofindividuals at risk or for immunization of the public in the case of anational emergency. Furthermore, even though recent work indicates thatdiluted stockpiled vaccine is still immunogenic, even at dilute doses(39,40), the use of the NYCBOH strain and bifurcated needle deliverytechniques are fraught with the problems of a replicating virus that canooze from pustules at the immunization site consequently posing apotential threat to immunocompromised individuals.

Vaccinia virus strains with changed virulence have been developed andprovide useful vectors for gene therapy and other applications [10]. Themost attenuated strain, MVA, has acquired multiple deletions andmutations through serial passage [11-14]. The virus can replicate inchick embryo fibroblasts, but it has a very limited mammalian host cellrange. Human cells are non-permissive for virus replication, which isblocked in the late stage of infection. The recommended mammalian hostcell is the baby hamster kidney 21 clone 13 (BHK21-CL13) cell line [15].

Many studies support the selection of MVA as a therapeutic orprophylactic vaccine in humans. The virus has been used effectively forprimary vaccination against smallpox in a test of 120,000 recipientvaccines [11]. When used as a vector, MVA has proved to be an efficientsystem because it expresses high levels of heterologous microbial andtumor antigen genes [13-21] in the absence of viral replication [18,21]. An added margin of safety has been demonstrated from use of thisstrain in animals and human clinical studies [22-26], includingimmunocompromised individuals [27-33]. Of particular relevance to oraldelivery are studies of Belyakov et al. [34] demonstrating strongmucosal and systemic immunity following intrarectal administration ofrecombinant MVA in a mouse vaccine therapy model.

In addition to safety and efficacy, oral immunization withreplication-deficient recombinant vaccinia virus such as MVA, offersmany other advantages over other vaccine candidates. The methodeffectively induces immune response in all three arms of the immunesystem, i.e., serum antibody, mucosal IgA antibody, and cell-mediatedimmunity. Compliance may be increased and overall costs reduced, becauseuse of an oral rather than a parenteral vaccine may enhance patient(and/or parental) acceptance and would obviate the need for syringes andneedles. Recombinant MVA can be constructed for multivalent vaccine oras a cocktail of MVA vaccines. Lyophilized vaccinia is extremelyheat-stable. Heating to 100° C. for two hours led to a loss of only onelog of infectivity, and storage at 45° C. for 2 years was still 100%successful in vaccination of volunteers [35]. These properties make oralMVA an ideal candidate vaccine.

Large-scale production of MVA is envisioned as a safe and immediatevaccination approach. Validation of vaccine safety and efficacy bybioassay on human intestinal cells is one method in the scope ofevaluation of MVA safety so that it meets pre-clinical and clinicaltesting standards for vaccine production. The human intestinal cells arenonpermissive for the virus, as are other human cells, but they produceviral antigens that are recognized by the host's immune effector cellsto stimulate systemic and mucosal immunity. A further benefit to the useof the intestine-derived cells as part of the testing and validationregimen is that it is well recognized that these types of epithelialcells can also act as antigen presenting cells in vivo. Thus, this is anexcellent approach to induce effective immunity.

REFERENCES

The following is a listing of patents pertaining to smallpox vaccine,vaccinia vectors, recombinant vaccinia virus, genetic (i.e., DNA)vaccines, other poxviruses as vectors, or vaccine therapies.

PATENT NO. TITLE 6,267,965 Recombinant poxvirus-cytomegaloviruscompositions and uses 6,265,189 Pox virus containing DNA encoding acytokine and/or a tumor associated antigen 6,265,183 Direct molecularcloning of foreign genes into poxviruses and methods for the preparationof recombinant proteins 6,165,460 Generation of immune responses toprostate-specific antigen (PSA) 6,103,244 Methods for generating immuneresponses employing modified vaccinia of fowlpox viruses 6,045,802Enhanced immune response to an antigen by a composition of a virusexpressing the antigen with a recombinant virus expressing animmunostimulatory molecule 5,997,878 Recombinantpoxvirus-cytomegalovirus, compositions and uses 5,989,561 Recombinantpoxvirus-calicivirus rabbit hemorrhagic disease virus (RHDV)compositions and uses 5,942,235 Recombinant poxvirus compositions andmethods of inducing immune responses 5,863,542 Recombinant attenuated ALV AC canaryopox virus containing heterologous HIV or SIV inserts5,858,373 Recombinant poxvirus-feline infectious peritionitis virus,compositions thereof and methods for making and using them 5,843,456Alvac poxvirus-rabies compositions and combination compositions and uses5,833,975 Canarypox virus expressing cytokine and/or tumor-associatedantigen DNA sequence 5,766,599 Trova fowl pox virus recombinantscomprising heterologous inserts 5,766,597 Malaria recombinant poxviruses5,762,938 Modified recombinant vaccinia virus and expression vectorsthereof 5,756,103 Alvac canarypox virus recombinants comprisingheterlogous inserts 5,756,102 Poxvirus-canine distemper virus (CDV)recombinants and compositions and methods employing the recombinants5,723,283 Method and composition for an early vaccine to protect againstboth common infectious diseases and chronic immune mediated disorders ortheir sequelae 5,691,449 Respiratory syncytial virus vaccines 5,688,920Nucleotide and amino acid sequences for canine herpesvirus GB, GC and GDand uses therefor 5,494,807 NYV AC vaccinia virus recombinantscomprising heterologous inserts 5,364,773 Genetically engineered vaccinestrain 5,348,741 Vector for recombinant poxvirus expressing rabies virusglycoprotein 5,294,548 Recombianant Hepatitis a virus 5,266,313 Raccoonpoxvirus as a gene expression and vaccine vector for genes of rabiesvirus and other organisms 5,262,177 Recombinant viruses encoding thehuman melanoma-associated antigen 5,223,254 Respiratory syncytial virus:vaccines 5,196,338 Recombinant vectors for Haemophilus influenzaepeptides and proteins 5,171,665 Monoclonal antibody to novel antigenassociated with human tumors 5,141,742 Vaccines against melanoma5,134,075 Monoclonal antibody to novel antigen associated with humantumors 5,110,908 Haemophilus influenzae peptides and proteins 5,108,744Vaccines for Haemophilus influenzae 5,098,997 Vaccines for Haemophilusinfluenzae 5,081,029 Methods of adoptive immunotherapy for treatment ofaids 5,068,106 t-PA solution of high concentration and use of thesolution in human and veterinary medicine 5,021,347 Recombinant vacciniavirus expressing E-protein of Japanese encephalitis virus 4,920,213Method and compositions useful in preventing equine influenza 4,877,612Immunological adjuvant and process for preparing the same,pharmaceutical compositions, and process 4,738,846 Vaccine for vesicularstomatitis virus 4,631,191 Methods and compositions useful in preventingequine influenza 4,603,122 Antiviral agent against herpes virusinfections 4,567,147 Attenuated smallpox vaccine strain 4,315,914Pharmaceutical compositions useful as cellular immunopotentiator andantitumor agent and process for production thereof 4,315,001 2-Deoxyglucose as an antiviral agent against herpes simplex 4,301,150 Method oftreating the clinical manifestations of viral diseases 4,218,436Compounds and methods 4,192,799 Conjugates formed by reacting aprostaglandin mimic compound with a carrier molecule 4,049,798 Methodfor the treatment of Herpes Simplex

In addition to the patents listed in this section, non-patentpublications cited in this application can be found in Section VI. (D)“Literature Cited” below.

SUMMARY OF THE INVENTION

The oral vaccine system and methods described herein use a live,defective vaccinia virus or a viral antigen preparation of such a virus,that can confer anti-smallpox immunity in the recipient. The inventionencompasses the combined methods by which the virus is grown using invitro cell culture methods (e.g., the methods of growing the I-MVA inthe baby hamster kidney cell line, BHK-21, followed by steps to purifythe virus, and quantitate the dosage), the characterization bioassaysfor its safety and efficacy prior to clinical use by oral delivery asper the immunization protocol, and the methods and components used forformulation. Cells used for vaccine preparation are derived fromINCELL's reference Master Cell Bank (MCB) and Working Cell Bank (WCB:n>200 vials) stocks. The MVA virus is propagated, for example, on BHK-21cells that are cultured to high culture density on microcarrier beads inplastic cell culture bags.

The vaccinia virus used for the vaccine can derive from the I-MVA strainor other defective vaccinia virus (DVV) strain incapable of generatinginfectious virus in a complete lytic cycle in human cells, but able toreplicate in an animal host cell which is permissive for the virus.

Safety, efficacy and potency components of the invention include invitro and immunoassays to evaluate the potential safety and potencyusing surrogate endpoint assays, such as infection of human intestinalcells, or other defined alimentary tract epithelial cells, and cellmediated immune (CMI) responses of cells from anti-vaccinia immunizedindividuals. The CMI responses can include bioassays for cytokines,cytotoxicity or other in vitro methods that reflect what would occur invivo.

The vaccine might be packaged in various forms, including packaging in aliquid, gel, or solid form that may be a tablet or gelcap or a componentof a food carrier material, such as a pudding or yogurt. In particularthe live vaccine would require packaging in a form that would allowdelivery into the human alimentary tract as whole virions that could betaken up in at the first part of the alimentary tract, i.e., the oralcavity, or at other sites, such as the intestine.

Various embodiments of the invention disclosed herein employ vacciniavirus, which was developed for gene therapy applications and has beenused as a vector to deliver genes, (e.g., tumor or microbial antigengenes), to the host as a live vaccine and carrying information intendedto confer immunity on the host by expression of the delivered gene.

The principle use of the oral vaccine system will be to protect againstpotential poxvirus infection, including smallpox, but incorporation ofother genes into the vaccinia virus vector is envisaged, such thatmulti-valent vaccine(s) against a variety of potential bio-agents,potential pathogens, or products of pathogens (e.g., toxins) can beprepared for oral delivery as disclosed herein. These can be packaged asseparate packages or may be in the same vector. They may be in a singlepackage or multiple packages, as another use of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. BHK-21 Cells Growing in Culture. This figure shows themorphology of normal BHK-21 clone 13 cells growing in monolayers. Thecells were fixed with Omnifix and stained with Giemsa.

FIG. 2. Immunoplaque Focus Formation Assay in BHK Cells to Titer Virus.This FIG. shows BHK cells with stained, I-MVA infected foci (arrows).Some stained individual cells are also seen, but they are not counted inthis assay.

FIG. 3. Serum Anti-MVA Antibody Titre in Rabbit Immunized byIntramuscular Route Measured by ELISA Assay. The rabbit was immunizedwith I-MVA in PBS combined with TiterMax Gold. ELISA was performed onMVA-coated microtiter plates and read at 405 nm after detection withPNPP colorimetric assay.

FIG. 4. Serum Anti-MVA Antibody Titre in Rabbit Immunized by theIntramuscular Route Measured by ELISA Assay. The rabbit was immunizedand the ELISA was performed as described above in FIG. 3.

FIG. 5. Serum Anti-MVA Antibody Titre in Sheep Immunized by theIntramuscular Route Measured by ELISA Assay. The sheep was immunizedwith I-MVA in PBS combined with TitreMax Gold. The ELISA was performedas described above in FIG. 3.

FIG. 6. ELISA Assays of Orally Immunized Rabbit Antibodies at EarlyImmunization Time Compared to Pre-bleed. The rabbit was immunized withI-MVA formulated for oral delivery. ELISA assays were performed asdescribed above in FIG. 3. The orally immunized rabbit producesdetectable anti-MVA antibodies by day 19 compared to the pre-bleed.

FIG. 7. ELISA Assays of Orally Immunized Rabbit Antibodies ShowContinued Antibody Production. An orally immunized rabbit continues toproduce anti-MVA antibodies, with slightly declining titers on day 69compared to day 46. ELISA was performed as described above in FIG. 3.

FIG. 8. ELISA Assays of Orally Immunized Sheep Antibodies. The sheen wasimmunized with I-MVA formulated for oral delivery. ELISA assays wereperformed as described above in FIG. 3.

FIG. 9. Comparative ELISA Assay of INCELL Antibody vs. CommercialAntibody. INCELL rabbit anti-MVA antibodies are equal to or better thancommercial antibodies and have a very high titer in serum and aspurified IgG when tested for binding to purified I-MVA in ELISA assays.ELISA assays were performed as described above in FIG. 3.

FIG. 10. Western Blot Assays with Rabbit Antibodies. Antibodies fromimmunized rabbits recognized purified I-MVA proteins. Purified I-MVA wasrun on gels, and separate lanes were tested for recognition of viralbands by the test antibodies indicated.

FIG. 11. Western Blot Assays with Sheep Antibodies. Purified MVA was runon gels and separate lanes were tested for recognition of viral bands bythe test antibodies indicated.

FIG. 12. Antibodies from Orally Vaccinated Animals Neutralize Virus.Sera from test animals used in immunoplaque assays showed infectiousI-MVA neutralization as measured by inhibition of plaque formationcompared to the controls (100%). Serum dilution is shown on the x-axis.Test animals: R3-37: Orally Immunized Rabbit 3 (day 37 pi): R4-37:Orally Immunized Rabbit 4 (day 37 pi): S2-19: Orally Immunized Shee 2(day 19 pi).

FIG. 13. MVA-Stimulation of DNA Synthesis in PBMCs. This figure showsdemonstrable stimulation of DNA synthesis in PBMCs from a sheenimmunized i.m. (“S-1”) and a sheep immunized orally (“S-2”).

FIG. 14. Complex Cellular and Humoral Immune Responses Elicited fromImmune Cells Stimulated with MVA. This is a diagram representing thecomplex humoral and cell-mediated immune responses, and examples of testparameters to assess immune responses against MVA, using in vitrosurrogate models to evaluate safety, efficacy and potency of the orallydelivered vaccine. The example is also applicable to other DVV and othertarget cells, effector cells, and infected cells. Similarly, the testmethods would be applicable to other orally delivered virus stocksintended to be ingested as a means of delivery of the vaccine. PBMCs:Peripheral blood mononuclear cells: SI: small intestine cells.

FIG. 15. The Manufacturing Approach: Closed Systems from VirusPropagation to Packaging. This is a diagram representing a systematicway to develop and manufacture vaccine product in a closed system usingavailable devices and clinical tools.

FIG. 16. The Manufacturing Approach: Closed and FDA Approved Devices.This is a diagram representing some examples of the types of devicesuseful for small-scale manufacturing. There are many other similardevices and larger scale options.

FIG. 17. The Manufacturing Approach: FDA Approved cGMP Components,Connectors, and Closed, Integrated Systems. Example of a closedisolation system with pumps, connectors, etc. that would compriseintegrated components that are easily configured and disposable.

FIG. 18. An Example of a Sample Pak for Oral Delivery. This is arepresentative sample of the types of packaging that might be used forunit dose delivery of the vaccine product as a tablet, a paste, or agel. There are many types of packaging and options such as blisterpacking that might be the preferred method.

DETAILED DESCRIPTION OF THE INVENTION

A. Manufacturing Methods and Materials

1. Facilities and Standards

All cells, virus and reagents are handled according to cGMP (currentGood Manufacturing Practice) standards. The manufacturing facility(anteroom, class 10,000 room, class 100 hoods) staff will use SOPs thatmeet FDA testing, validation, and QA/QC manufacturing standards. Thesemeasures are taken to accelerate the process from discovery to product.

2. The BHK-21 Clone 13 (BHK21-CL13} Cell Line

The BHK-21 CL 13 cell line (ATCC #CCL-10) is used as the permissivecells to propagate the stock virus. The rationale for choosing this lineis that it is permissive for DVV such as MVA and is easily grown inculture. An example showing BHK-21 cells growing in culture is shown inFIG. 1. Cells are maintained as recommended by the ATCC in a modifiedMinimal Essential Medium with Earle's Salts [EMEM], 0.1 mM non-essentialamino acids, and 1 mM sodium pyruvate (e.g., GIBCO or other vendor) with10% v/v fetal bovine serum (FBS; e.g., Hyclone or other vendor), oranother suitable growth medium. They are subcultured using 0.25%trypsin, 0.03% EDTA (GIBCO) at subcultivation ratios of 1:2 to 1:10.Cells used for vaccine preparation are derived from INCELL's referenceMaster Cell Bank (MCB) and Working Cell Bank (WCB; n≧200 vials) stocks.The banked cells have been checked for sterility by standard microbialgrowth and mycoplasma PCR assays of the MCB and WCB and characterized byDNA fingerprinting to assure identity.

3. Virus Propagation and Analyses

The INCELL propagated strain of MVA (ATCC #VR01508), designated I-MVA,has been routinely propagated by standard methods and titered by thepreferred method of immunoplaque assay as detailed below. Otherquantitative methods that have been used include either end pointdilution in BHK-21 cells to obtain a 50% tissue culture infectious dose(TCID50/ml) or IU (infectious units), as detailed by Dresden et al.[15]. For in vitro and in vivo assays, virus has been purified byultracentrifugation through a 36% sucrose cushion using standard viruspurification methods. The BHK-21 cells are grown in culture (also termed“in vitro”) and infected at 0.1 FFU per cell to generate large lots ofvirus harvested at 72+/−2 hours post infection (p.i). The BHK-21 cellcultures can be monolayers in various types of bioreactor or scale-upcultures, including culture flasks, stacked systems, culturemicrocarrier beads, or other appropriate substrates to culture thecells. The resultant virus stocks can be concentrated or purified byultracentrifugation, ultrafiltration, or other standard methods, thentitered on BHK-21 cells and stored for packaging. Part of the stock isaliquoted for use in the bioassays and for QA testing. All lotinformation is entered into the master database and inventory managementsystem which were developed as part of the invention and its use.

For immunoplaque assays, BHK-21 cells are seeded at a density of 4.5×10⁴cells per well of a 24-well plate in growth medium (EMEM, 10% FBS, plusadditives, as described above). After overnight attachment, when thecells are 80-90% confluent, they are infected with I-MVA by mixing thevirus with EMEM prepared as per the growth medium but with 2% ratherthan 10% FBS (=EMEM:2 infectivity medium). Test I-MVA source materialsare generally diluted in 10⁻³ to 10⁻⁷ for cell culture derived,unconcentrated supernatants, and 10⁻⁶ to 10⁻¹⁰ for gradientcentrifugation purified or otherwise concentrated (e.g., ultrafiltered)virus. Dilutions are made in EMEM:2 infectivity medium. Cells thatreceive no virus but are otherwise incubated with EMEM:2 infectivitymedium and treated the same are used as negative controls. Referencevirus stocks that are known to produce 100-200 plaques per well areincluded in the assays as positive controls to assure performance of theassay. Cultures are gently swirled to assure even virus distributionthen incubated for 24 hr at 37° C., in a 5% CO₂, 95% air environment. Atthe end of the incubation period, the medium is removed from the wells,the cells are fixed with 0.5 ml 1:1 acetone:methanol for 5 min, thefixative is removed and 1 ml CMF-PBS is added to each well. The rinsesolution is removed and anti-vaccinia virus primary antibodies (e.g.,rabbit, sheep, human or other source) and the biotin or otherchromagen-labeled secondary antibodies used at an effective dilution(e.g., 1:500 to 1:1000) to easily visualize the immunoplaques. For moststudies, 1:500 dilutions of each of the primary (rabbit anti-vaccinia;Accurate Chemical or INCELL-prepared) and secondary (HRP anti-rabbitIgG; SIGMA or other vendor) antibodies were used. An example showingBHK-21 cells and the appearance of plaques in the immunoplaque assay isshown in FIG. 2.

B. Antiviral Antibodies and Applications

1. Intramuscular Depot Immunization with TiterMax Gold

For rabbit immunizations, 10⁸ FFU in 0.5 ml PBS were combined with 0.5ml TiterMax Gold using a double hub emulsification needle (push antigeninto TiterMax first, aqueous into oil phase) for mixing. The emulsionwas injected into 4 sites (0.2 ml each) over both shoulders and bothhind quadriceps. For sheep immunizations, 2×10⁸ FFU in 1 ml PBS werecombined with 1 ml TiterMax Gold as above and inject 0.4 ml twice intoeach hind quadriceps. Animals were bled periodically to test antibodyproduction. Good antibody titers are present within 4-6 weeks and remainhigh for several months.

2. Oral Immunization Formulae and Methods

A variety of oral immunization formulae can be used for immunization.Oral immunization is done by preparing a formula in which the virusremains viable (as determined by infectivity of released virus from theorally delivered paste and separate components of the paste formulaelisted below) and is captured in nanoparticles and micelles as part ofthe protective formulation that includes aqueous and oil-basedcomponents, as well as suspending agents and carriers that protect thevirus from degradation and allow it to be absorbed from the oral cavityand the intestine.

As an example of the formulation used for the the studies shown in theFigures, virus is prepared (at 10⁸ per rabbit or 2×10⁸ per sheep) bymixing virus in a solution of Hetastarch (hydroxyethyl starch, clinicalgrade; 6% w/v; Baxter), 40% (v/v) mannitol [UPS grade higher; SIGMA orother vendor], 0.15% (v/v) AAFA™ (nutritional supplement grade fish oil;INCELL 5% (v/v) glycerol (UPS grade; SIGMA or other vendor), 0.5% (w/v)gelatin (SIGMA) at a volume that will achieve a final concentration of5×10⁴ to 2×10⁸ infectious FFU, depending on the effective or test doseexpected (e.g., 10⁶ to 10⁸ for humans, depending on immunizationstatus). In the animal studies, doses were at 10⁸ per rabbit and 2×10⁸per sheep. Gel-sol virus carrier (GSVC) excipient components wereprepared as an equal mixture (1:1:1; Avicel® CE-15(microcrystallinecellulose and guar gum), Avicel® 591 (water-dispersible microcrystallinecellulose containing sodium carboxymethylcellulose (NaCMC) andAc-Di-Sol® (internally-crosslinked, water insoluble sodiumcarboxymethylcellulose (NaCMC)) [source of all components: FMCProducts]) which was slowly added (with vortexing) to a finalconcentration of 10% (w/v).

Taste-testing (humans and animals) revealed that the formulae waspalatable as a slightly sweet paste-gel type of formulae that caused noaftertaste and which could be subsequently dried (e.g., for tableting)and still maintain infectious virus as measured by infection ofdissolved materials on BHK-21 cells after they had been dried and storedfor various time periods, supporting long-term storage as a tablet orpaste-gel material that maintains biological activity.

C. Bioassays and Biochemical Methods for Safety, Efficacy and Potency

A variety of bioassays and biochemical analyses are done to evaluate thevaccine. These include: (a) human cell line nonpermissiveness withexpression of vaccine antigens (a safety test); (b) viral antigenexpression and production compared to previous lots and referencestandards (i.e., potency); and (c) activation of humoral andcell-mediated immunity (e.g., potency and efficacy) in infected animals.These are imperative types of assays to evaluate each virus lot and theoverall potential variability between lots of virus.

1. Safety and Potency Bioassays: I-MVA Infection of Human Cells in vitro

INCELL has the only long-term continuous cell lines derived from humanintestine (HI). As part of the pre-clinical testing, the HI cells werebe grown in M3:10™ medium (INCELL) as monolayer cultures using standardmethods so that they maintained functional cell and organ-specificmarkers that make them useful in vitro surrogates for orallyadministered products, including vaccines or drugs. Master and WorkingCell Banks of these cells were banked in the INCELL repository prior toinitiating these studies.

As part of the evaluation of I-MVA lots of oral vaccine, the HI testcell line(s) lines were seeded into culture vessels in M3:10™ (INCELL)growth medium, allowed to attach, then infected with test lots of virusessentially as described above for the FFU immunoplaque assays or asdetailed elsewhere (15) for alternate cell infectivity studies. For eachset, parallel cultures of uninfected and infected permissive BHK-21 cellcontrols, and dilutions of prepared reference virus, were tested tovalidate the bioactivity of the virus stocks.

An example of the study showing comparative infectivity of I-MVA forhuman intestinal and other human cells compared to the permissive BHK-21cells are shown in Table 1. The important vaccine safety-relatedconclusion from the results shown in this table is that the I-MVA strainused to prepare the vaccine does not grow in human cells but readilyreplicates in the permissive BHK-21 cells.

TABLE 1 Safety Assays: I-MVA Does Not Replicate in Human CellsDescription of Cell Line 48 hr p.i. Test Groups* Designation VirusTiter** Starting Inoculum NA: virus only   1 × 10⁵ Positive ControlBHK-21 1.3 × 10⁷ Negative Control NA: media + virus only 4.3 × 10⁴Description of Cell Line 48 hr p.i. Human Cells Tested Designation VirusTiter Normal Duodenum HUD 00818 <10⁵ Normal Duodenum HUD 00919 <10⁵Normal Jejunum INJE 00510a <10⁵ Normal Jejunum INJE 00526a <10⁵ NormalJejunum INJE 00729 <10⁵ Normal Ileum INIL 00510a <10⁵ Normal Ileum INIL00729 <10⁵ Normal Colon NCM 356 <10⁵ Normal Colon NCM 425 <10⁵ NormalColon NCM 460 <10⁵ Normal Colon CSC-1 <10⁵ Colon Cancer CaCo2 <10⁵ ColonCancer Colo 205 <10⁵ Normal Dermis HSK 740DF <10⁵ *Cells seeded asmonolayers, Infected with virus (MOIca. 0.1) At 48 hrs p.i.,immunoplaque assays were done to determine titer. **Titer shown refersto the FFU/ml of each cell line. Note that the virus replicated in thepermissive BHK cells but not in any of the human cell lines.

2. Immunoassays to Evaluate Antibodies and Antigens

Three methods are used to evaluate production of the viral antigens andanti-viral antibodies as a measure of potency of the lots produced: (a)ELISA assays, (b) Western blots, and (c) Immunocytology.

a. ELISA Assays

ELISA plate assays are done to quantitate the amount of anti-virusantibodies produced against the virus or the amount of virus antigenproduced by infected cells. Such assays have many variables andmethodologies. An example test method is as follows. The virus stocksare diluted to 0.1 to 4 μg/ml in carbonate buffer and coated onto ELISA96-well plates at 25 μl/well for 4 hours to overnight at 37° C. and thenwashed 3 times with PBS-Tween40 (PBS-T). The antigen-coated plates areblocked with 3% BSA at 200 μl/ml for 1 hr at 37° C. (on a rockerplatform) and washed 3 times with PBS-T. Virus reference test antigensand reference antibody dilutions are used at known positiveconcentrations and ratios as positive controls. Test antibodies arebracketed for assay at multiple dilutions, based on expected ranges, inreplicates of N=4, with test dilution samples added to the plates at 25μl/well. After incubation for 2 hr at 37° C., the plates are washed withPBS-T. All comparative values are analyzed using INCELL's customizedplate analysis software in concert with statistical and graphicsprograms.

Importantly, antibodies have been consistently demonstrable in all ofthe immunized animals. In general the orally immunized animals had asomewhat lower titer than the animals that received intramuscular depotimmunization with the TiterMax. However, as shown below, the circulatingantibody titer did not necessarily correlate with neutralizationdifferences and CMI may actually be higher in the orally immunizedanimals.

b. Western Blots

For Western blot analyses, cell protein lysates were prepared andresolved by electrophoresis on a SDS-8% polyacrylamide gel, thentransferred onto nitrocellulose for 2 h in a buffer containing 25 mMTris, 192 mM glycine, and 20% methanol (pH 8.6). The blots were blockedovernight at 4° C. in a PBS blocking buffer containing 1% BSA and 0.1%NP40 and then incubated for 1 h at room temperature with rabbit or sheepanti-vaccinia antibody diluted 100-fold in blocking buffer. After beingwashed with 0.1% NP40 in PBS, the blots were incubated for 1 h at roomtemperature with goat anti-rabbit or anti-sheep IgG light (Amersham)diluted 1000-fold in blocking buffer, washed again, and exposed to X-rayfilm for comparative evaluation and image analysis to quantify thesamples.

As shown in the examples of Western blots in FIGS. 10 and 11, theimmunized rabbits and sheep were able to elicit antibodies that couldrecognize viral proteins on a Western blot analysis. This furtherverifies the specificity of the antibodies for the virus, as wasdemonstrated with sera from all of the test animals.

c. Immunocytology to Visualize for Antigen Expression by Infected Cells

Immunocytology assays may have many variables and methodologies. Anexample test method is as follows. To analyze cells for visualizing theexpression of viral or cell antigens, the cells are grown as monolayers.This is done on multi-well plates or on Lab-Tek (Corning) slides orattached to coated slides by standard cytocentrifugation protocols. Forimmunodetection assays, standard protocols have primary antibody dilutedin PBS to an optimal working dilution followed by incubation with thecells that have been fixed in Omnifix or another antigen appropriatefixative.

For immunocytology assays, the cells are fixed with 10% formalin (Sigma)for 1 hr at 4° C. and blocked with 3% bovine serum albumin (BSA; Sigma)in calcium- and magnesium-free phosphate buffered saline with 0.01%Tween-20 (CMF-PBS-T). Specific anti-vaccinia polyclonal rabbit antibody(Accurate Chemical) or newly derived antibodies are added to the fixedand rinsed cells. Cells are stained according to the general proceduresdetailed in the Vectastain® Elite ABC Kit by the manufacturer (VectorLaboratories). Briefly, each sample is incubated with test antibody atthe appropriate working dilution of the antibody followed by abiotinylated goat anti-rabbit or anti-mouse secondary antibody (Sigma:1:2000). The samples are quenched with 0.3% hydrogen peroxide anddeveloped with a combination of an avidin-linked peroxidase conjugateand the 3,3′-diaminobenzidine (DAB) chromagen. The slides arecounterstained with hematoxylin (Biomeda Corp.), mounted with aqueousmounting medium (Biomeda Corp), and visualized with a Nikon Microscope.Photographs are taken using a digitized format and photo capturesoftware. The stained cells look similar to the individual stained cellsshown in FIG. 2 of the immunoplaque assay. When such assays are doneusing antibodies from i.m. or orally immunized animals, theimmunostained cells look similar.

3. Demonstration of Neutralizing Antibody and Protection in ImmunizedAnimals

For immunoplaque reduction or neutralization assays, the methods are thesame for immunoplaque assays through the set-up step, but the virusinoculum is pre-incubated for at least 1 hr with serum or purified IgGprior to adding the virus-antibody inoculum. Otherwise, the remainingsteps of the protocol are they same. When the virus is pre-incubatedwith the test serum containing antibody the incubation step is at 37 Cand the serum is usually heat-inactivated at 56 C for 30 min prior toincubation with the virus.

FIG. 12 shows that orally immunized animals could neutralize infectiousI-MVA as measured by inhibition of plaque formation compared to thecontrols (100%). Similar results were obtained with i.m. immunizedanimals. It was concluded that all orally immunized animals producedneutralizing antibody. In the example, rabbits showed a stronger effectbut sera were collected 37 days pi vs. only 19 days pi for the sheep.

This work complements studies done with mice in which orally immunizedmice are challenged with an infectious vaccinia strain, such as WR, andthe immunized animals are protected from the associate morbidity andmortality of the challenge virus.

4. Assessing Potency with Cell-Mediated Immunity Assays

a. Cells from Immunized Donors

The Peripheral Blood Mononuclear Cells (PBMCs) are obtained fromperipheral blood and separated using standard methods. Either freshPBMCs or pre-qualified (known responder) PBMCs from cryopreservation areused for testing. Cell separation methods and cell-mediated immunityassays may have many variables and methodologies as described below.

One example method is to take PBMCs from immunized donors and determinewhether or not they can respond to stimulation with the immunizingantigen, in this case, I-MVA. To that end, PBMCs from a sheep immunizedintramuscularly (i.e., “Sheep 1”) and a sheep immunized orally (i.e.,“Sheep 2”) were added to RPMI culture medium containing 10% (v/v)autologous plasma. Quadruplicate cultures of 10⁵ cells per well of a96-well plate with or without MVA antigen, or control wells withoutcells, were compared to assess a cellular response to MVA antigen asmeasured by stimulation of DNA synthesis using a BRDU ELISA-based assayas detailed below. Results of an example study are shown in FIG. 13,where it is clear that both sheep had demonstrable cell stimulation. Theconclusion form these studies is that oral immunization can effectivelyinduce cell-mediated immunity against I-MVA and,thus, presumably againsta related invading poxvirus, such as smallpox.

Another example method is as follows. The collected blood cells orcultured cells are layered over warm Histopaque and centrifuged at 400×gto separate dead from viable cells. The number and viability of the cellpopulation is assayed by standard 0.25% Trypan Blue Dye Exclusion usingINCELL's SOP. On day zero, 5×10⁶ viable cells are seeded into 6-wellplates containing a final volume of 5 ml MR:20™ (INCELL) or othersuitable culture medium. After an overnight culture adaptation period, asubset of PBMCs are infected with I-MVA at an MOI of 1 TCID/cell for 2 hand the remaining autologus PBMCs are readied for co-culture asdescribed below. After washing twice, 8×10⁵ I-MVA-infected PBMCs areirradiated or treated with mitomycin C (25 mcg/ml) so they can no longerdivide. For CMI activation studies, the infected cells are then added to5×10⁶ autologous PBMCs, which had also been cultured overnight. Aftersubsequent culture for 4-7 days, DNA synthesis is determined by addingradiolabeled ³H-thymidine or 5-Bromo-2-deoxyuridine (BRDU) for the final4 to 18 hr of culture period to measure incorporation into DNA. The³H-thymidine is measured by liquid scintillation counting oftrichloroacetic acid [TCA] (10%)-precipitated cellular DNA added toscintillation fluid and counted. The BRDU is measured using an ELISAassay and highly specific anti-BRDU antibody linked to a chromagen(e.g., biotin). Control cultures (cells and media only) are grown at thesame time but do not receive I-MVA or I-MVA infected cells. The controlvalues are either subtracted from the test assays with infected cells togive specific incorporation numbers or they are used to determine therelative labeling index by the equation (DNA[t]-DNA[c])/DNA[c]).

To amplify the response or to initiate a de novo response, cultures canbe re-stimulated weekly using freshly prepared I-MVA infected andautologous PBMCs at a responder to stimulator ratio of 2:1 andsupplemented with 25 IU/ml IL-2. After four cycles of re-stimulation,bulk cultures can be further tested for immune activation and many testparameters. Controls include purified virus and uninfected cells.Comparative test parameters of immune activation include cytokineproduction, cytotoxicity against target cells, and cell activation(including target cell death and mixed lymphocyte reaction [MLR]) usingthe methods described in more detail below. For these studies, ANOVA isused to statistically compare the groups.

b. Cytokine Assays

Inflammatory mediators or cytokines (e.g., TNF-α; IFN-gamma) generatedby PBMCs after in vitro stimulation with I-MVA, I-MVA-infected cells, orno (control) stimulus. Cells are assayed by immunoassays of culturesupernatants from multi-well plates or by ELISPOT assays in which cellsare attached to plates containing an antibody against the cytokine ofinterest (e.g., IFN-gamma). Supernatants from stimulated PBMCs (infectedcells or purified virus) are compared to control cultures (i.e., mediaonly and uninfected cells with no stimulus) ELISA, Western or dot-blotassays can be used to compare cytokine production by cells in the testgroups. Values of stimulated cells are adjusted for background andbaseline values of the control groups so that induced or increasedcytokine production is measured.

Cytotoxic T lymphocytes (CTLs) are also tested for their production ofTNF-α or IFN-gamma following co-culture with selected lines ofI-MVA-infected compared to uninfected cells. Cytokine assays may havemany variables and methodologies. An example test method is as follows.Stimulator cells are infected for 4 hr with I-MVA at an MOI of 1FFU/cell, extensively washed, and plated in 6-well plates at 8×10⁵cells/well. After an overnight incubation 12-15 hr at 37° C., Effector Tcells (5×10⁶ cells/well) are added. Effector cells co-cultured withuninfected cells are used as negative reference controls. All assays aredone at least in triplicate. For cytokine (e.g., TNF-α or IFN-gamma)assays, supernatants are harvested after 40 h, and the cytokine content(ng/ml) is determined by multi-well plate ELISA or ELISPOT assays (e.g.,R&D Systems; Cell Systems).

c. Mixed Lymphocyte Reaction (MLR)

MLR assays may have many variables and methodologies. An example testmethod is as follows. “Responder” PBMCs (1×10⁵ cells/well) are seededinto 96-well culture plates. I-MVA-infected autologous PBMCs (1-3 hrpi), and mock-infected control cells are treated with mitomycin C (25ug/ml) then added (1×10⁵ cells/well) to the responder cells (1:1 ratio).Cell proliferation is measured after 96 hrs with a BRDU ELISA assay asdescribed above.

d. Chromium Release Assays (CRAs)

CRAs may have many variables and methodologies. An example test methodis as follows. The lytic activity of either in vitro-stimulated“Effector” {E} Cytotoxic T Lymphocytes (CTLs) are tested againstI-MVA-infected or uninfected Target {T} cells in a 4-hr standard ⁵¹Crrelease assay. Target cells are infected for 2 h with I-MVA at an MOI of1 FFU/cell, washed once, then labeled with 100 μCi Na⁵¹CrO₄ for 1 h at37° C. After 4 washes with PBS, labeled target cells are plated inU-bottomed 96-well plates at 1×10⁴ cells/well and incubated at 37° C. At15-18 hr after infection, effector cells are incubated with the targetcells at various E: T ratios (0:1, 25:1, 50:1, 100:1). After 4 hr, theplates are centrifuged to pellet the cells, 100 μl of supernatant perwell is collected, counted, and recorded as Mean+/−SD counts per minute(cpm) of replicate samples (n=4). The specific ⁵¹Cr release isdetermined by subtracting the background counts of cells in the 0:1{E:T} group where there are no effector cells added. Results of the testgroups are compared by ANOVA statistical analyses to determinedifferences between groups at a P value <0.05.

D. Manufacturing

The manufacturing steps for production of the vaccine will include theuse of existing disposable cell propagation devices, connectors, andother closed system technologies that are adaptable from laboratory cellculture to scale-up manufacturing of large batches.

FIG. 15 is an example of the overall manufacturing approach from viruspropagation to packaging. In this example, the MVA virus is propagatedon BHK-21 cells that are cultured to high culture density onmicrocarrier beads in plastic cell culture bags, followed byconcentration and purification of the virus, combining the virus with aproprietary oral delivery formulation as the “vaccine mixture”,processing the vaccine mixture to a tablet, paste, gel, liquid or otheroral delivery form, then packaging it in a foil package, blister pack,or other standard form as a single unit dose. All procedures andmaterials for virus propagation and handling at all steps of themanufacturing are selected with the notion that they can be scaled upfrom laboratory lots to 200 or more liters, then discarded after use, toremove the validation and other aspects related to cleaning andsterilization of vessels and other manufacturing components.

FIG. 16 shows examples of the types of closed and FDA approved,disposable products that will be obtained from qualified vendors andused for manufacturing steps. They include a variety of plasticwaredisposables that can be scaled larger manufacturing needs. FIG. 17 showsan example of how FDA Approved cGMP Components, Connectors, and Closed,Integrated Systems might be combined as a manufacturing step.

FIG. 18 shows an example of an Oravax™ sample package prepared as a unitdose package for oral delivery. The unit dose would include I-MVA in aformulation that has been tableted or is prepared as a paste or gel thatcan be squeezed from the foil or other packaging. The package can bemade as foil or blister packages to which tablets are added or it can bemade as a form, fill, seal method whereby the package is formed, filledwith the vaccine product (gel, paste, liquid, then sealed in a packagethat can be opened for single use consumption. Features of the packageare that it does not allow light or moisture and, preferably, theproduct can be stored at room temperature or refrigerated, but does notrequire freezer temperatures to maintain stability.

Manufacturing approvals and outcomes include evaluation of product:safety, potency, efficacy, stability, shelf life and other measures thatinclude innovative and unique elements for this application. Safety ofthe virus lots to be used in the oral delivery formulation is tested byusing appropriate human target cells, i.e., normal human cells frommultiple donors and several regions of the alimentary tract. Potency ismeasured by determining that the vaccine virus lot is infectious forpermissive cells with a quantified titer as determined by immunoplaqueor other assay. Efficacy is measured by the production of protectiveimmunity, such as virus neutralization with associated lack ofinfectivity for the host target cells or animals, following oraldelivery of the vaccine with the immunizing virus strain included in aformulation that protects the virus and augments immune responsiveness,and has stability and a shelf life of at least a year, with preferablestorage at room temperature.

Said formulation may include gelatin, cellulose, or a variety of otherexcipients as ingredients, or the formulation may be a gel or a foodcarrier such as a pudding or similar formulation that would include thevirus as a component. As another embodiment flavorings, emulsifiers, orother additives may be included in the formulation of the product, thedelivery vehicle or other components of the packaged material. The I-MVAimmunogen can be packaged as a solution, as single doses, as a paste orgel, or in a food or nutritional substance in a plastic container,pillow-pack, tear-pack, straw tube packaging or other suitable packagingfor the liquid, gel or food carrier formulation.

D. Literature Cited

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1. An oral vaccine comprising: a replication-defective or deficientvaccinia virus, or a modified vaccinia virus strain that is unable togenerate infectious virus in human cells but is able to replicate in ananimal host cell which is permissive for the virus, in a formulationcomprising hydroxyethyl starch, mannitol, nutritional supplement gradefish oil, glycerol, and gelatin; wherein gel-sol virus carrier excipientcomponents comprising a mixture of equal parts of microcrystallinecellulose and guar gum; water-dispersible microcrystalline cellulosecontaining sodium carboxymethylcellulose (NaCMC); and internallycross-linked, water-insoluble sodium carboxymethylcellulose (NaCMC) isadded to the formulation to form a paste or gel for oral delivery. 2.The oral vaccine of claim 1, wherein the formulation comprises 6% (w/v)hydroxyethyl starch, 40% (v/v) mannitol, 0.15% (v/v) nutritionalsupplement grade fish oil, 5% (v/v) glycerol, 0.5% (w/v) gelatin.
 3. Theoral vaccine of claim 2, wherein the gel-sol virus carrier excipientcomponents are added to said formulation to final concentration of 10%(w/v).
 4. The oral vaccine of claim 1, wherein the strain of virus isMVA.
 5. The vaccine of claim 1, wherein said virus is provided in therange of 5×10⁴ to 2×10⁸ infectious FFU.
 6. The vaccine of claim 5,wherein said virus is provided in a range of 1×10⁶ to 1×10⁸ infectiousFFU.
 7. The vaccine of claim 1, wherein the vaccine is packaged in aunit dose form.
 8. A formulation for oral delivery of a vaccinia virusto immunize an animal or human subject, comprising hydroxyethyl starch,mannitol, nutritional supplement grade fish oil, glycerol, and gelatin,wherein a mixture of equal parts of microcrystalline cellulose and guargum; water-dispersible microcrystalline cellulose containing sodiumcarboxymethylcellulose (NaCMC); and internally cross-linked,water-insoluble sodium carboxymethylcellulose (NaCMC) is added to theformulation to form a paste or gel for oral delivery.
 9. The formulationof claim 8, comprising 6% (w/v) hydroxyethyl starch, 40% (v/v) mannitol,0.15% (v/v) nutritional supplement grade fish oil, 5% (v/v) glycerol,0.5% (w/v) gelatin, wherein a mixture of equal parts of microcrystallinecellulose and guar gum; water-dispersible microcrystalline cellulosecontaining sodium carboxymethylcellulose (NaCMC); and internallycross-linked, water-insoluble sodium carboxymethylcellulose (NaCMC) isadded to said formulation to a final concentration of 10% (w/v).
 10. Amethod for immunizing a mammal against vaccinia virus, comprising orallyadministering an effective dose of a replication-defective or deficientvaccinia virus, or a modified vaccinia virus strain that is unable togenerate infectious virus in human cells but is able to replicate in ananimal host cell which is permissive for the virus, in a paste or gelformulation comprising hydroxyethyl starch, mannitol, nutritionalsupplement grade fish oil, glycerol, and gelatin, wherein a mixture ofequal parts of microcrystalline and guar gum; water-dispersiblemicrocrystalline cellulose containing sodium carboxymethylcellulose(NaCMC); and internally cross-linked, water-insoluble sodiumcarboxymethylcellulose (NaCMC) is added to the formulation to form apaste or gel for oral delivery.
 11. The method of claim 10, wherein saidformulation comprises 6% (w/v) hydroxyethyl starch, 40% (v/v) mannitol0.15% (v/v) nutritional supplement grade fish oil, 5% (v/v) glycerol,0.5% (w/v) gelatin, and wherein a mixture of equal parts ofmicrocrystalline cellulose and guar gum; water-dispersiblemicrocrystalline cellulose containing sodium carboxymethylcellulose(NaCMC); and internally cross-linked, water-insoluble sodium,carboxymethylcellulose (NaCMC) is added to said formulation to finalconcentration of 10% (w/v).
 12. The method of claim 10, wherein saidvirus is delivered into the alimentary tract as whole virions that aretaken up at multiple sites in the tract and into the host circulation tostimulate host immunity.
 13. The method of claim 12, wherein two of saidsites are the oral cavity and the small intestine.
 14. The method ofclaim 10, wherein the immune response is a systemic response.
 15. Themethod of claim 10, wherein the immune response is a mucosal response.16. The method of claim 10, wherein the immune response includes humoraland cell-mediated immunity.
 17. The method of claim 10, wherein both asystemic and mucosal response is produced.
 18. The method of claim 10,wherein said virus is provided in a range of 5×10⁴ to 2×10⁸ infectiousFFU.
 19. The method of claim 18, wherein said virus is provided in arange of 1×10⁶ to 1×10⁸ infectious FFU.
 20. The method of claim 10,wherein said strain of virus is MVA.
 21. The method of claim 10, whereinin the formulation is dried and delivered in tablet form.
 22. The methodof claim 10, further comprising one or more booster immunizations. 23.An oral vaccine comprising: a replication-defective or deficientvaccinia virus, or a modified virus that is unable to generateinfectious virus in human cells but is able to replicate in an animalhost cell which is permissive for the virus, in a formulation comprisinghydroxyethyl starch, mannitol, nutritional supplement grade fish oil,glycerol, and gelatin, wherein gel-sol virus carrier excipientcomponents comprising a mixture of equal parts of microcrystallinecellulose and guar gum; water-dispersible microcrystalline cellulosecontaining sodium carboxymethylcellulose (NaCMC); and internallycross-linked, water-insoluble sodium carboxymethylcellulose (NaCMC) isadded to the formulation to form a paste or gel for oral delivery; andwherein said vaccine is effective in protecting an animal against alethal challenge with an infectious strain of vaccinia virus.
 24. Anoral vaccine comprising: 5×10⁴ to 2×10⁸ infectious FFU of MVA in aformulation comprising 6% (w/v) hydroxyethyl starch, 40% (v/v) mannitol,0.15% (v/v) nutritional supplement grade fish oil, 5% (v/v) glycerol,0.5% (w/v) gelatin, and wherein a mixture of equal parts ofmicrocrystalline cellulose and guar gum; water-dispersiblemicrocrystalline cellulose containing sodium carboxymethylcellulose(NaCMC); and internally cross-linked, water-insoluble sodiumcarboxymethylcellulose (NaCMC) is added to said formulation to finalconcentration of 10% (w/v).
 25. The oral vaccine of claim 24, wherein1×10⁶ to 1×10⁸ infectious FFU of MVA are present in said vaccineformulation.